PHYTOCHEMICAL ANAYSIS AND DETERMINATION OF ANTIOXIDANT

PROPERTY OF Elaeagnus triflora

An Undergraduate Research
Presented to the Pharmacy and Medical Technology Department
Centro Escolar University
City of Malolos

In Partial Fulfillment
of the Requirements for the Degree
Bachelor of Science in Pharmacy

by
Cudia, Catherine Joy J.
Cuevas, Lery B.
Espino, Kevin C.
Gonzales, Maegan Shaira E.
Manahan, Irish Camille C.

April 2019
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CHAPTER 1
THE PROBLEM AND ITS SETTING

Introduction

Filipinos are now becoming more aware of the risks of living an unguided life

without medical intervention or advising, however with the rising technological age, people

are becoming more open to knowledge being a new trend in our society, and is now more

aware of the risks and consequences that could develop, and with this kind of awareness,

people are now finding ways to improve and promote healthy living.

Along with other risk factors that lead to serious or even worse, one of the issues

that should be considered are deadly diseases that deteriorate normal life as we know it,

and one example of those major factors that cause multiple diseases and symptoms are free

radicals or also known as “Reactive Oxygen Species” (ROS), and until now, we are still

finding ways on how to reduce the said effects of free radicals with antioxidants (Di Matteo

V, Esposito E, et. al. 2003)

Antioxidants help prevent or stop cell damage caused by oxidants (Breene, 2016).

As the name implies, antioxidants are substances that are capable of counteracting the

damaging, but normal, effects of the physiological process of oxidation in animal tissue.

Antioxidants are nutrients (vitamins and minerals) as well as enzymes (proteins in your

body that assist in chemical reactions). They are believed to play a role in preventing the

development of such chronic diseases as cancer, heart disease, stroke, Alzheimer's disease,

Rheumatoid arthritis, and cataracts. Oxidative stress occurs when the production
 2

of harmful molecules called free radicals is beyond the protective capability of the

antioxidant defenses. Free radicals are chemically active atoms or molecular fragments that

have a charge due to an excess or deficient number of electrons (Parnes, 2002).

Free radicals and oxidants play a dual role as both toxic and beneficial compounds,

since they can be either harmful or helpful to the body. They are produced either from

normal cell metabolisms in situ or from external sources (pollution, cigarette smoke,

radiation, medication). When an overload of free radicals cannot gradually be destroyed,

their accumulation in the body generates a phenomenon called oxidative stress. This

process plays a major part in the development of chronic and degenerative illness such as

cancer, autoimmune disorders, aging, cataract, rheumatoid arthritis, cardiovascular and

neurodegenerative diseases (Lien, 2008)

As time goes by, lot of new compounds are being discovered with promising

antioxidant activity, but some undiscovered sources are starting to run out, including plants

and animals which has the potential to have anti-oxidant property and to cure illnesses

cause by free radicals according to Hail N (2008). A number of medicinal plants have been

subjected to detailed chemical investigations and this has led to the isolation of pure

bioactive molecules which have been pharmacologically evaluated. As a result, new drugs

have been discovered, along with new applications.

The plant kingdom represents an enormous reservoir of biologically active

compounds with various chemical structures and protective/disease preventive properties

(phytochemicals). These phytochemicals, often secondary metabolites present in smaller

quantities in higher plants, include the alkaloids, steroids, flavonoids, terpenoids, tannins,
 3

and many others. Nearly 50% of drugs used in medicine are of plant origin, and only a

small fraction of plants with medicinal activity has been assayed. There is therefore much

current research devoted to the phytochemical investigation of higher plants which have

ethnobotanical information associated with them. The phytochemicals isolated are then

screened for different types of biological activity (Harborne, 1998).

The plant investigated in the current study was Elaeagnus triflora Roxb., from

the Elaeagnaceae family also known as “Lingaro” in the Philippines. There is only limited

information about this plant for it is presently understudied ethnomedicinal plant. The

researchers aimed to make use of the all the parts available to undergo phytochemical

analysis and determining its antioxidant activity.

Background of the Study

The E.triflora plant was one of the listed ethnomedicinal plants used by traditional

healers therapeutically in Laguna, Philippines from the study of Fiscal (2017) which was

utilized in treating stomachache, headache, muscle pain, urinary tract infection, liver

disease, gall bladder stone, without prior knowledge or scientific basis on the chemical

constituents it possess.

E.triflora is a climbing shrub with long branches which are covered with minute,

brown scales. Leaves are entire, subelliptic to ovately oblong, 4 to 9 centimeters long, 2 to

4 centimeters wide, pointed at both ends or blunt at the base, shining and dark green above,

and coppery or sometimes grayish-white beneath. Flowers are yellow and fragrant,

occurring singly in the axils of the leaves. Fruit is oval, about 1.5 to 3 centimeters long,

pale red or pinkish, sweet and juicy when ripe (Stuart, 2016). Located
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in thickets and forests at low and medium altitudes, ascending to 1,500 meters throughout

the Philippines. It can also be found in Taiwan, Indonesia, Malaysia, New Guinea, and

Australia. The plant is sometimes cultivated as an ornamental or hedge plant, or for its

edible fruit. Fruit can be eaten raw. According to Folkloric the ripe fruit are given to

children suffering with amoebic dysentery and fruits as astringent. The curiosity of

determining the potential medicinal use of the plant was the factor that driven the

researchers to conduct a study by performing phytochemical analysis and determining the

possible antioxidant property of the plant. As it being an understudied plant, the discovery

of its medicinal properties and uses can serve as valuable information in future drug

researches and innovation.

Conceptual Framework

The phytochemical research approach is considered effective in discovering

bioactive profile of plants of therapeutic importance (Masih, 2012). Plants are able to

produce a large number of diverse bioactive compounds. High concentrations of

phytochemicals, which may protect against free radical damage, accumulate in fruits and

vegetables (Suffredini, 2004). Plants containing beneficial phytochemicals may

supplement the needs of the human body by acting as natural antioxidants (Boots, 2008).

Various studies have shown that plants are rich source of antioxidants. For instance,

vitamins A, C, E, and phenolic compounds such as flavonoids, tannins, and lignins, found

in plants, all act as antioxidants (Suffredin, 2004). The consumption of fruits and

vegetables has been linked with several health benefits, a result of medicinal properties and

high nutritional value (Valko, 2006).

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In this study the researchers determined the phytochemicals present in the various

extracts obtained from different plant parts of Elaeagnus triflora. Further, the antioxidant

property of the various extract was evaluated, through this effort, Elaeagnus triflora’s

potential health benefits may be revealed. This study will performed based on the

schematic diagram shown in Fig. 1.

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Collection of plant sample

Drying of plant sample

Sequential extraction

Extractive

Phytochemical Antioxidant
Physicochemical Evaluation
Screening
Flavonoids
Alkaloids
Terpenoids (2, 2-diphenyl-1-
Glycosides picrylhydrazyl)
Phenols DPPH Assay
Amino acids Organoleptic
Saponins Test Solubility Test Fourier
Tannins Appearance 5% NaHCO3 Transform
Carbohydrates Color 5% NaOH Infrared Ferric Reducing
5% HCl Spectroscopy Antioxidant
Lipids Odor Power)
Proteins Distilled water (FTIR)
FRAP Assay
Quinines
Oxylates
Anthraquinones
Xanthoproteins
Phlobatamins
Resins

Figure 1. Showing the step/ process in the conduct of the study of Phytochemical
Analysis and Determination of Antioxidant Property of Elaeagnus triflora
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Statement of the Problem

This study aims to identify the different phytochemical constituents and determine

the antioxidant property of the various extract from Elaeagnus triflora Roxb.

Specifically, the study seeks to answer the following questions:

1. What is the percentage yield of extractives from the various extracts of Elaeagnus
triflora?

2. What are the phytoconstituents present in the various extract of Elaeagnus

triflora ?

3. What are the physio-chemical characteristics of the various extracts from

Elaeagnus triflora when subjected to:

3.1 Organoleptic Evaluation

3.2 Solubility Test

3.3 FTIR Analysis

4. Which among the various extracts from Elaeagnus triflora exhibited the best
antioxidant activity?

Significance of the Study

Antioxidants are very sought for nowadays, ranging from synthetic materials up to

natural and even rare organic materials, and these effects are very much available in

varieties but are still limited in perception due to lack of human knowledge on the said

effects or not bothering to go in depth with the subject.

The importance of this study is for us to know what possibilities are available with

the search for more antioxidant property carrying compounds and materials in order to

prove that there is much more to search for in this field, and to make people realize
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that antioxidants could be used in a way that could be helpful in their lives with no doubt

on using it even if the said compound has a side effect.

Patient. The patients will be informed about the importance of removing toxins in

their body that can help them improve from their health problems; example is the liver

problems, which are commonly related with antioxidants.

Community. People will give importance on how they take good care the plant

simply on their own house since they will be informed about how it will help to remove

toxins around it and purifies air which is the number one benefit of this plant at home.

Manufacturing firm. Manufacturing Company will have ideas that Elaeagnus

triflora has an antioxidant property which they can used in the future to develop different

dosage form.

Researcher. Future researcher can obtain ideas about the proposed study, and it

can also serve as there reference when they conduct study related to the topic.
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Scope and Delimitations

This study is limited to the determination of phytochemicals and antioxidant

analysis of the crude extracts obtained from leaves, stem and bark of Elaeagnus triflora

Roxb.

This study made use of sequential extraction of constituents from leaves, stem, and

bark of E.triflora using n-Hexane, Ethyl acetate, methanol and 50% methanol as

menstruum. The various fractions were subjected to phytochemical analysis. The

physicochemical characteristics of the constituents such as organoleptic, solubility test,

FTIR analysis were determined.

The study is limited only to the determination of antioxidant property of each

fraction determination via in-vitro assays i.e. DPPH and FRAP. No in-vivo analysis was

conducted as well as ALD50 and other toxicity studies due to time and resources

limitations. Factors such as climate, soil, harvest time and other environmental factors will

be excluded as possible source of variance.

Definition of Terms

The following terms are hereby defined for better understanding of the present

study.

Alkaloids. Refers to any of a class of nitrogenous organic compounds of plant

origin that have pronounced physiological actions on humans. They include many drugs

(morphine, quinine) and poisons (atropine, strychnine).

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Antioxidant. This refers to man-made or natural substances that may prevent or

delay some types of cell damage.

Bioactive. Pertains to a substance having a biological effect

Free radicals. This pertains to an atom or molecule that bears an unpaired electron

and is extremely reactive, capable of engaging in rapid chain reactions that destabilize other

molecule.

Flavonoids. It refers to a group of phytonutrients (plant chemicals) found in almost

all fruits and vegetables. Along with carotenoids, they are responsible for the vivid colors

in fruits and vegetables.

Oxidation. This process pertains to a chemical reaction that transfer electrons or

hydrogen from a substance to an oxidizing agent.

Phytochemicals. This are non-nutritive plant chemicals that have protective or

disease preventive properties. They are non-essential nutrients, meaning that they are not

required by the human body for sustaining life.

Phytonutrients. This is a substance found in certain plants which is believed to be

beneficial to human health and help prevent various diseases.

Secondary metabolites. It refers to chemicals produced by plants for which no role

has yet been found in growth, photosynthesis, reproduction, or other "primary" functions.
 11

Steroids. This is any of a large class of organic compounds with a characteristic

molecular structure containing four rings of carbon atoms (three six-membered and one

five). They include many hormones, alkaloids, and vitamins.

Tannins. It is a yellow or brown chemical that is found in plants such as tea. It is

used in the process of making leather and in dyeing.

Terpenoids. These are any of a large class of organic compounds including

terpenes, diterpenes, and sesquiterpenes. They have unsaturated molecules composed of

linked isoprene units, generally having the formula (C5H8) n.

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CHAPTER 2

Review of Related Literature and Studies

This chapter presents the related literature and studies after the thorough and in-

depth search done by the researchers. The information from foreign and local sources

relevant in the study are gathered to give clarification and lay the foundation of this

research work.

Mohammad Hosein Farzaei’s (2015) study “A comprehensive review on

phytochemical and pharmacological aspects of Elaeagnus angustifolia L.” reveals that

various in-vitro experimental studies confirmed significant antioxidant potential of E.

angustifolia. Okmen and Turkcan reported that flavonoids isolated from leaf methanolic

extract have remarkable antioxidant properties. In-vitro investigation showed that ABTS

(2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) scavenging effect of leaf methanol

extract (100 mg/ml) was 84% by ABTS decolorization assay. Various fractions,

particularly hydroethanolic fraction, obtained from the fruits demonstrated radical

scavenging activity. Wang et al. reported that flavonoid glycosides, quercetin 3,4’-O-βD-

diglucoside, isorhamnetin-3-O-β-D-galactopyranoside, quercetin 3-O-β-D-

galactopyranoside- 4’-O- β-Dglucopyranoside and isorhamnetin 3-O-β-D-

galactopyranoside-4’-O-β-D-glucopyranoside from fruits possess antioxidant property

concentration-dependently in term of ABTS+ radicals scavenging, DPPH (1,1-diphenyl-

2picrylhydrazine) radical scavenging and superoxide anion radical scavenging method.

Ethanolic extract of the flowers possesses total antioxidant activity in ferric thiocyanate

assay, total reducing ability through Fe3+- Fe 2+ transformation assay, DPPH and
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superoxide anion radical scavenging activity, as well as suppression of lipid peroxidation.

Bucur et al. reported that the pharmaceutical formulations, dermatological preparation and

soft extract from flowers and young branches possess antioxidant activity, and showed that

the antioxidant activity of flowers soft extracts (antioxidant range 43.6–45.9%) was higher

than young branches (range 32.7– 43%) in chemiluminescence assay. It seems that this

capacity is attributed to flavonoids and polyphenols constituents. The antioxidant activity

of the extracts showed linear relationship with polyphenols but non-linear dependence with

flavonoids.

Graziele Francine Franco Mancarz’s study (2016) entitled “Antimicrobial and

Antioxidant Activity of the Leaves, Bark and Stems of Liquidambar styraciflua L.

(Altingiaceae).” The butanol fraction of the bark and stem showed the strongest antioxidant

activity, which was stronger than those of the reference standards that were used in this

study. The value displayed by the ethyl acetate fraction of this same plant organ represented

the second strongest antioxidant effect; however, the average did not differ from those of

the ascorbic acid standard and the ethyl acetate fraction of the leaves. The antioxidant

activity found for the crude extract of the leaves was statistically similar to that exhibited

by the gallic acid and rutin standards. From the results obtained using the

phosphomolybdenum methodology, it can be seen that for both the samples and the

reference patterns, the total antioxidant capacity is dose-dependent, with the highest

concentration tested here (250 µg/mL) corresponding to the highest total antioxidant

capacity of all of the analyzed solutions.

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There are numerous studies that prove that since these diseases are mediated by

oxidative stress and disbalance between pro-oxidant and antioxidant factors, antioxidants

may play a pivotal role in preventing or slowing the progression of these conditions.

Antioxidants help to decrease the chances of developing these diseases such as Heart

disease. It is speculated that a critical step in development of atherosclerosis is oxidation

of low-density lipoprotein (LDL) (a type of bad cholesterol in blood) within the arterial

wall. Several studies show an association between low intakes of dietary antioxidants to an

increased frequency of heart disease. (Mandal, 2014).

Antioxidants exert their protective effect in cancer by decreasing oxidative damage

to DNA and decreasing abnormal increases in cell division. Pro-oxidants, or those who

generate free radicals, stimulate cell division and these form the beginnings of mutagenesis

and tumor formation. When a cell with a damaged DNA strand divides, it gives rise to

disturbed and deformed clusters of cells that form the cancer. Recent studies suggest that

free radicals may be involved in the development of pulmonary disorders such as asthma.

Antioxidants have been seen to reduce the development of asthmatic symptoms. Vitamin

C, vitamin E, and beta carotene supplementation has been associated with improved lung

function. Free radicals can also damage nerves and the brain. Neural tissue may be

particularly susceptible to oxidative damage. This is because the brain receives a

disproportionately large percentage of oxygen and has large amounts of polyunsaturated

fatty acids which are highly prone to oxidation and oxidative damage. Diseases implicated

to oxidative stress include Alzheimer's disease, Parkinson's disease and dementia.

 15

Kelly (2012) expressed her concept that a considerable interest has risen in the idea

that oxidative stress is instrumental in the etiology of numerous human diseases. Oxidative

stress can arise through the increased production of reactive oxygen species (ROS) and/or

because of a deficiency of antioxidant defenses. Antioxidant deficiencies can develop as a

result of decreased antioxidant intake (such as vitamins C and E), synthesis of enzymes

(such as superoxide dismutase and glutathione peroxidase) or increased antioxidant

utilization. Insufficient antioxidant enzyme synthesis may in turn be due to decreased

micronutrient availability (such as selenium, magnese, copper and zinc). Of those diseases

linked with oxidative stress, cardiovascular disease provides the strongest evidence for the

protective role of antioxidants. A high consumption of fruit and vegetables, which are good

sources of antioxidants, is associated with a lower coronary risk. More specifically, there

is evidence of a reduced coronary risk in populations with high blood levels of the

antioxidant nutrients, vitamins C and E. Evidence is also accumulating that diabetes, and

microvascular complications associated with diabetes, involve oxidative stress and have

compromised antioxidant status. In addition, patients who develop acute respiratory

distress syndrome (ARDS) also exhibit clear evidence of oxidative stress. Definitive proof

for active oxygen formation and oxidative cell damage being causative rather than a result

of other underlying these pathologies remains elusive; however, evidence is sufficiently

compelling to suggest that antioxidants are potential therapeutic agents in the above

conditions.

According to the article entitled “Antioxidants: What you need to know”, copyright

by American Academy of Family Physicians (AAFP). Antioxidants are

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chemicals that help stop or limit damage caused by free radicals, defines by an article

“Antioxidants: What you need to know”, copyright by American Academy of Family

Physicians. Your body uses antioxidants to balance free radicals. This keeps them from

causing damage to other cells. Antioxidants can protect and reverse some of the damage.

They also boost your immunity. You can get most of these antioxidants by eating a healthy

diet. This includes a mix of colorful fruits and vegetables. Whole grains, seeds, and nuts

also provide good nutrients. You can get Vitamin A in milk, butter, eggs, and liver.

Vitamin C is in most fruits and vegetables. Eat fruits such as berries, oranges, kiwis,

cantaloupes, and papayas. Eat vegetables such as broccoli, bell peppers, tomatoes,

cauliflower, Brussels sprouts, and kale. Vitamin E is in some nuts and seeds. For example,

almonds, sunflower seeds, hazelnuts, and peanuts. You can find it in green leafy vegetables

such as spinach and kale. You also can find it in soybean, sunflower, corn, and canola oils.

Beta-carotene is in brightly colored fruits and vegetables. Eat fruits such as peaches,

apricots, papayas, mangoes, and cantaloupes. Eat vegetables such as carrots, peas, broccoli,

squash, and sweet potatoes. It also is in some leafy green vegetables such as beet greens,

spinach, and kale. Lycopene is in pink and red fruits and vegetables. This includes pink

grapefruits, watermelon, apricots, and tomatoes. Lutein is in green leafy vegetables such

as spinach, collards, and kale. You also can find it in broccoli, corn, peas, papayas, and

oranges. Selenium is in pasta, bread, and grains, including corn, wheat, and rice. You can

find it in animal products, like beef, fish, turkey, and chicken. You also can find it in nuts,

legumes, eggs, and cheese.

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From the study entitled “Native Fruit Species in the Philippines and Their

Phenotypic Traits and Potential Uses” by Pablito M. Magdalita et al. (2016) Alingaro

(Eleaegnus triflora Roxb.) is native to the Philippines, found in Taiwan, Australia,

Malaysia and New Guinea. Dark green leaves are simple and elliptic, ripe oblong fruits are

dark red, fruit weight is 1.3 ± 0.95 g, red flesh is soft, sub-acid and juicy. Ripe fruits are

eaten raw which is also used to treat amoebic dysentery.

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CHAPTER 3

Methods and Procedures

This chapter focuses on the method and procedures utilized to investigate the active

components and antioxidant property of the plant parts of Elaeagnus triflora from the

family of Elaeagnaceae.

Setting of the study

The lingaro plant scientifically known as Elaeagnus triflora Roxb. were collected

from Mabacan, Calauan, Laguna during the month of July 2018. The plant sample was

authenticated at the UP Institute of Biology, College of Science UP Diliman, Manila. The

experimental procedures from the phytochemical analysis for antioxidant assay were

conducted at Centro Escolar University Science Laboratory Malolos City, Bulacan. The

FTIR, DPPH assay and FRAP assay were conducted at UP Institute of Chemistry, College

of Science UP Diliman, Manila.

Method of Research

The researchers made use of the experimental method of research, wherein

systematic laboratory procedures and laboratory instruments were used to identify the

active constituents of the plant and knowledge that are required in the study. The Elaeagnus

triflora was subjected to physical and phytochemical evaluation:

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1. Determination of percentage yield of extractives from the various extracts of

(Elaegnus triflora)

1.1 Collection and preparation of E.triflora

The parts were collected during the months of July 2018 at RC Conservation

Farm, Mabacan, Laguna. The leaves, stem, and bark were carefully washed

with tap water prior to air drying. The dried parts were ground using Vesmach

RT-N12 Pulvurizer 450G Pulverizing Machine.

1.2 Extraction

The powdered leaves, stem and bark from E.triflora were subjected to

sequential extraction using four different solvents of increasing polarity for

about 48hours at room temperature in the following order: n-hexane, ethyl

acetate, methanol, and 50% aqueous methanol, then it was filtered. This

process was repeated in three series in each menstruum and plant part as shown

in Figure 2 (Kumar,2012).

1.3 Computation of the Percentage Yield

About 100 grams of the sample was weighed and placed in a suitable

container, the menstruum was added to the dried sample using a 1:10 ratio of

sample:menstruum. These underwent evaporation using the rotary evaporator

and residue was weighed and computed using the formula:

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒
% Yield = 𝑥 100
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑝𝑙𝑎𝑛𝑡 𝑝𝑎𝑟𝑡𝑠
 20

Extraction with n-Hexane (thrice) for 48 h at 30 C

Marc n-Hexane extract

Extraction with Ethyl acetate (thrice) for 48 h at 30 C

Marc Ethyl acetate extract

Extraction with Methanol (thrice) for 48 h at 30 C

Marc Methanol extract

Extraction with 50% Aqueous Methanol (thrice) for 48 h at 30 C

Marc 50% Aqueous Methanol extract

Figure 2 Sequential extraction process of Elaeagnus triflora’s leaves, stem, and bark
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2.1 Phytochemical Evaluation of the Elaeagnus triflora tree parts

2.1.1 Phytochemical Evaluation

The filtrate underwent partial evaporation using Heidolph Rotary

Evaporator Heating bath Hei-VAP (EU), at 687oC for 30 minutes, ethyl acetate

at 77.1oC for 90 minutes, methanol at 64.7 oC for 3 hours and lastly 50%

aqueous methanol at 65oC for 4 hours at a speed of 760 torr for every 2 liters

of filtrate.

2.2 Test for alkaloid analysis

About two milliliters of plant extract were dissolved with eight milliliters

1% HCl, was warmed, filtered, and then subjected to following test:

2.2.1 Mayer’s Test

About two milliliters of filtrate was treated with two drops of Mayer’s reagent

by the side of the test tube. A white or creamy precipitate indicates positive

result.

2.2.2 Wagner’s Test

About two milliliters of filtrate was treated with two drops of Wagner’s

reagent by the side of the test tube. A reddish-brown precipitate indicates

positive result.

2.2.3 Dragendorff’s Test

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About two milliliters of filtrate was treated with two drops of Dragendorff’s

reagent by the side of the test tube. A red precipitate indicates positive

result.

2.3 Test for Xanthoproteins

2.3.1 Test for Quinines

About two milliliters of extract was treated with one milliliter of

alcoholic KOH solution. Appearance of colours ranging from red to blue

indicates positive result.

2.3.2 Test for Oxylates

About two milliliters of extract was added to two milliliters acetic acid

containing one drop of FeCl3. Two milliliters H2SO4 was added to it. Brown

ring at interface indicates positive result.

2.4 Test for Glycosides

2.4.1 Keller-Killiani Test

About two milliliters of the extract diluted to five milliliters in water,

two milliliters of glacial acetic acid containing one drop of ferric chloride

solution was added. This was underlayed with 1 milliliter of concentrated

H2SO4. A brown ring at the interface indicated the presence of a

deoxysugar characteristic of cardenolides. A violet ring may appear

 23

below the brown ring, while in the acetic acid layer a greenish ring may

form just above the brown ring and gradually spread throughout this layer.

2.5 Test for Carbohydrates

2.5.1 Molisch Test

About two milliliters of plant extract was treated with two drops of

alpha naphthol solution. Formation of violet ring junction indicates

presence of carbohydrates.

2.5.2 Fehling’s Test

About two milliliters of Fehling’s solution was boiled (2.5 milliliters

each of Fehling A and B) in a test tube, two milliliters of plant extract were

added and boiled. Formation of brick red or red precipitate indicates presence

of reducing sugars.

2.6 Test for Terpenoids

2.6.1. Salkowski’s Test

About two milliliters of the plant extract was dissolved in two

milliliters chloroform and then two milliliters of concentrated H2SO4 was

added from the sides of the test tube. The test tube was shaken for a few

minutes. Red colour development in the chloroform layer indicated the

presence of sterols.

2.6.2 Test Liebermann-Burchard Test

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About two milliliters extract was dissolved with two milliliters

of chloroform and filtered. The filtrates were treated with three drops

of acetic anhydride, boiled and cooled. About one milliliters of

concentrated H2SO4 was added. Formation of brown ring junction

indicates the presence of terpenoids.

2.7 Test for Phenol

2.7.1 Ferric Chloride Test

About two milliliters of test sample of each extract was taken

separately in water, warmed and filtered. To a small volume of this filtrate,

a few drops of 5 % w/v solution of FeCl3 prepared in 90 % alcohol were

added. Appearance of a dark green or deep blue colour indicated the

presence of tannins.

2.8 Test for Saponins

About two milliliters of extract was boiled with five milliliters distilled water.

To the filtrate, about three milliliters of distilled water was further added and shaken

vigorously for five minutes. Frothing about one centimeter layer indicates positive

results.

2.9 Test for Anthraquinones

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About two milliliters of the extract was boiled with ten milliliters of H2SO4

and filtered while hot. The filtrate was shaken with five milliliters chloroform. The

chloroform layer was pipetted into another test tube and 1ml of dilute ammonia was

added. The resulting solution was observed for color changes.

2.10 Test for Phlobatannins

About 0.8 grams per milliliters of plant extract was boiled with 1% aqueous

HCl, the deposition of red precipitate indicates presence of phlobatannins.

2.11 Test for Resins

About two milliliters extract, three to four milliliters of CuSO4 solution

was added separately and the tubes were shaken vigorously for one to two minutes

the resulting solution was allowed to separate. Formation of green color

precipitate indicated the presence of resins.

2.12 Test for Flavonoid

2.12.1 Ferric Chloride Test

About two milliliters of extract was dissolved in two milliliters of distilled

water. To this two drops of 10% FeCl3 was added. A dark green colour indicates

presence of phenolic compounds

2.13 Flavonoidal Test

 26

About two milliliters of 1% NH3 was added to 0.5 milliliters extract and

then one milliliters concentrated was added. Appearance of yellow color indicated

positive result.

2.14 Test for Proteins

2.14.1 Millon’s Test

About two milliliters of extract were treated with ten drops of

Millon’s reagent and placed in a boiling water. A flesh color is a positive

result.

2.14.2 Xanthoproteic Test

About two milliliters of extract were treated with three drops of

concentrated HNO3. Formation of yellow color indicates presence of

proteins.

2.15 Test for Lipids

2.15.1 Stain Test

About two milliliters of equivalent plant sample was added with

ten milliliters petroleum ether or hexane. A white paper was prepared and

placed two drops of petroleum ether extract. If there is permanent greasy

stain it is positive.

3 Physicochemical Evaluation of the Extract

 27

3.1 Organoleptic Test

The extract was observed for its color, taste, odor and appearance.

3.2 Solubility Test

For the test in solubility, the researchers used different solvents

ranging from basic to acidic mediums, a small amount of the plant extract

was used and placed in different test tubes for testing, there were different

solvents in each test tube, Distilled water, 5%NaHCO3, 5%NaOH, 5%HCl

M.Jones and S.A. Fleming, et.al (date). This aqueous solubility test is

effective for determining the presence or absence of acidic or basic functional

groups present in the plant sample.

3.3 Solubility in Water

About five milligram of plant extract was dissolved in a one milliliter

of distilled water and was tested using litmus paper. If the red litmus paper

turns to blue, the sample contains basic functional groups (presence of amine).

If blue litmus paper turns to red, the sample contains acidic functional group

(presence of carboxylic acid), if the plant sample is soluble in water, there is

no need for another solubility test using the other solvent since these solvent or

predominantly water and the plant sample will dissolve in them as well.

3.4 Solubility in aqueous Sodium Hydroxide

 28

About five milligrams of plant extract was dissolved in a one

milliliter of 5% NaOH. If the plant sample is soluble, meaning it contain

acidic functional group (presence of carboxylic acid and phenol).

3.5 Solubility test in aqueous Sodium Bicarbonate

About five milligrams of plant extract was dissolved in a one

milliliter 5% sodium bicarbonate, if the sample is soluble, it contain acidic

functional group (presence of carboxylic acid) because sodium bicarbonate is

a good solvent for determining acidic functional group containing pka <8.

3.6 Solubility in aqueous Hydrochloric acid

About five milligrams drops of plant extract was dissolved in a one

milliliter of 5% HCl. If the sample is soluble, it contain basic functional group

(presence of amine).

4 Determination of Antioxidant Activity of the various extract

Reagents/ Materials

4.1 Preparation of Vitamin C as Positive Controls

Equivalent amount of different doses of positive controls were

weighed. Ascorbic acid was used as the positive control, researchers made f5,

10, 20, 50, and 100mcg/ml of ascorbic acid by dissolving fifty milligrams in

ethanol to make concentration of 1mg/ml (100mg%).

 29

4.2 Antioxidant Assay

Antioxidant property of the plant extracts from the leaves, stem, and

bark extract of E.triflora was determined using DPPH assay and FRAP

Assay. The researcher used 5mcg/ml, 10mcg/ml, 20mcg/ml, 30mcg/ml,

50mcg/ml, and 100mcg/ml concentration in performing DPPH assay and

FRAP assay.

4.3 2,2-diphenyl-1-picrylhydrazyl Assay (DPPH Assay)

DPPH radical scavenging activity of crude was measured. Extract

solutions were prepared by dissolving 0.05 grams of dry extract in fifty

milliliters of methanol. An aliquot of two millilietrs 0.004% DPPH solution

in methanol was mixed with one milliliters of plant extracts in methanol at

five different concentrations; five, ten, twenty, fifty, and one hundred

micrograms per milliliter (5mcg/ml 10mcg/ml, 20mcg/ml, 50mcg/ml, and

100mcg/ml) and incubated at 25oC for 30 minutes. The absorbance of the

text mixture was read at 517 nanometer using a spectrophotometer against

DPPH control containing only one milliliter of methanol in place of the

extract. All experiments were prepared thrice along with Ascorbic acid

(standard). The percentage inhibition was calculated using the following

expression:

𝐴 blank −𝐴 sample
% Inhibition = X 100
𝐴 blank
 30

Where, blank and sample stand for the absorption of the blank and absorption of

the tested extract solution respectively.

4.4 Ferric ion reducing antioxidant power (FRAP Assay)

The reducing antioxidant power of the extracts was determined by the

method the method of Oyaizu. The researcher used different concentration:

ten, twenty, thirty, fifty and one hundred micrograms per milliliter

(10mcg/ml, 20mcg/ml, 30mcg/ml, 50mcg/ml, and 100mcg/ml) in 1ml of

distilled water were mixed with phosphate buffer (3.0ml, 0.2M, pH 6.6) and

K4Fe(CN)6·3H2O (2.5ml, 1%). The mixture was incubated in an incubator at

50oC for 20 minutes. After incubation, 2.5ml of C2HCl3O2 (10%) was added

to the mixture and centrifuged for 10 minutes at 3000 rpm. The upper layer

of solution (2.5ml) was mixed with distilled water (2.5ml) and FeCl3 (0.5 ml,

0.1 %). The samples absorbance was read at 700 nanometer against a blank

using UV-spectrophotometer. Ascorbic acid was used as a standard.

The percent FRAP scavenging of both plant extracts from leaves, stem

and bark of E.triflora and standard compounds were calculated:

(𝐴c−𝐴s)
%Scavenged [FRAP] = [ }𝑥 100
𝐴c

Where Ac is the absorbance of the control and As is the absorbance in

the presence of the antioxidant property from the plant extracts of the leaves,

stem, and bark extract of E.triflora.

 31

5 Instrumental evaluation for Antioxidant using Spectrophotometer

The crude extract was subjected to FTIR to determine the specific

functional group present in the leaves extract of E.triflora. Three to five

milligrams of sample were placed directly in IR plates with potassium KBr as

solvent. The assembled fixed thickness was ascended on the sample holder of

the instrument and closed the lid. The mixture was pressed using a screw and

was inserted in the FTIR machine. The rendered spectrum was shown on the

screen.
 32

CHAPTER 4

Presentation, Analysis and Interpretation of Data

This chapter presents the results obtained from the preliminary screening, physical

and chemical evaluation and instrumental testing of extracts based on the methods and

procedures used in this study. The data collected were also examined and interpreted.

1. The Percentage (%) yield of extractives from various extracts of Elaeagnus triflora

Table 1
Percentage yield of various extractives
Plant Sample N-Hexane Ethyl acetate Methanol 50% Aqueous Methanol
Leaves 9.54% 5.21% 13.15% 6.69%
Stem 7.37% 7.29% 10.42% 4.41%
Bark 7.91% 7.39% 9.66% 10.22%

The dried leaves, stem and bark of Elaeagnus triflora were macerated with [1]

n-Hexane, [2] Ethyl acetate, [3] Methanol and [4] 50% Aqueous Methanol chronologically,

evaporated to syrupy consistency and was weighed to get the percentage yield. The

percentage yield of [1] n-hexane,; (a) Leaves was 9.54 % (b) Stem was 7.37% (c) Bark

was 7.91% [2] Ethyl acetate; (a) Leaves was 5.21% (b) Stem was 7.29% (c) Bark was 7.39

% [3] Methanol; (a) Leaves was 13.15% (b) Stem was 10.42% (c) Bark was 9.66% [4]

(50%) Aqueous Methanol (a) Leaves was 6.69% (b) Stem was 4.41% (c) Bark was 10.22%.

2. Phytoconstituents present in the various extract of Elaeagnus triflora

Table 2
Phytochemical Screening Results of various crude extracts under series of solvent
performed in Elaeagnus triflora of the Leaves
ID TEST N-Hexane Ethyl acetate Methanol 50% Methanol
 33

Alkaloids
Mayer’s Test +++ + + ++
Wagner’s Test +++ + ++ ++
Dragendorff’s Test +++ ++ + ++
Xanthoproteins - - + -
Quinines - - - -
Oxylates + + + -
Glycosides
Keller-Killiani’s Test + + + ++
Carbohydrates
Molisch’s Test - - - -
Fehling’s Test - - + +
Terpenoids
Salkowski’s Test - + + ++
Liebermann-Burchard + + + ++
Test
Phenols
Ferric Chloride Test - - - -
Saponins
Foam Test - +++ + -
Anthraquinones + ++ + +
Phlobatannins - - - -
Resins - - - +
Flavonoids
Ferric Chloride Test - - - -
Test for Flavonoids - - - -
Proteins
Millon’s Test - - - -
Xanthoproteic Test - - - -
Lipids
Stain Test - - - -
Tannins
1% Ferric Chloride Test - - - -
Gelatin Test + - - -
Test for Tannins - + - -
Note: (+++) Strong Presence, (++) Moderate Presence, (+) Present, (-) Absent

According to the results in the leaf sample of Elaeagnus triflora, the positive result

indicates the presence of the following constituents in various extractives: Terpenoids*,

Alkaloids, Glycosides, Anthraquinones are present in all solvents. Terpenoids are present

in Ethyl acetate, Methanol, 50% Aqueous Methanol. Proteins are present in Ethyl acetate.

Saponins are present in Ethyl acetate and Methanol.

Table 3
Phytochemical Screening Results of various crude extracts under series of solvent
performed in Elaeagnus triflora of the Stem
ID TEST N-Hexane Ethyl acetate Methanol 50% Methanol
 34

Alkaloids
Mayer’s Test +++ + - ++
Wagner’s Test +++ ++ - ++
Dragendorff’s Test +++ ++ - ++
Xanthoproteins - - - -
Quinines - - - -
Oxylates + ++ + +
Glycosides
Keller-Killiani’s Test - ++ + ++
Carbohydrates
Molisch’s Test - - - -
Fehling’s Test - - + ++
Terpenoids
Salkowski’s Test + - ++ +
Liebermann-Burchard Test + + ++ +
Phenols
Ferric Chloride Test - - - -
Xanthoproteic Test - - - -
Saponins
Foam Test - + ++ +
Anthraquinones - + + +
Phlobatannins - - - -
Resins - - - -
Flavonoids
Ferric Chloride Test - - - -
Test for Flavonoids - - - -
Proteins
Millon’s Test - - - -
Xanthoproteic Test - - - -
Lipids
Stain Test - - - -
Tannins
1% Ferric Chloride Test - - - -
Gelatin Test - - - -
Test for Tannins - - - ++
Note: (+++) Strong Presence, (++) Moderate Presence, (+) Present, (-) Absent

Based on the phytochemical analysis done using the stem sample of Elaeagnus

triflora, the positive results indicates the presence of the following constituents in various

extractives: Terpenoids*and Oxylates are present in all solvents. Alkaloids are present in

N-Hexane, Ethyl acetate and 50% Aqueous Methanol. Terpenoids are present in

N-Hexane, Methanol and 50% Aqueous Methanol. Glycosides, Lipids and

Saponins are present in Ethyl acetate, Methanol and 50% Aqueous Methanol. Tannins are

present only in 50% Aqueous Methanol.

 35

Table 4
Phytochemical Screening Results of various crude extracts under series of solvent
performed in Elaeagnus triflora of the Bark
ID TEST N-Hexane Ethyl acetate Methanol 50% Methanol
Alkaloids
Mayer’s Test +++ +++ + -
Wagner’s Test +++ +++ + -
Dragendorff’s Test +++ +++ + -
Xanthoproteins - - + -
Quinines - - - -
Oxylates - ++ + -
Glycosides
Keller-Killiani’s Test + + + +
Carbohydrates
Molisch’s Test - - - -
Fehling’s Test - - + +
Terpenoids
Salkowski’s Test + + + +
Liebermann-Burchard + + ++ +
Test
Phenols
Ferric Chloride Test - - + -
Saponins
Foam Test + +++ + +
Anthraquinones + ++ ++ +
Phlobatannins - - - -
Resins + - - -
Flavonoids
Ferric Chloride Test - - + -
Test for Flavonoids - - - -
Proteins
Millon’s Test + + - +
Xanthoproteic Test - - - -
Lipids
Stain Test - - - -
Tannins
1% Ferric Chloride Test - - - -
Gelatin Test - - - -
Test for Tannins - + + -
Note: (+++) Strong Presence, (++) Moderate Presence, (+) Present, (-) Absent

The results from the stem sample of Elaeagnus triflora, the positive results are

indicating the presence of the following constituents in various extractives: Terpenoids,

Glycosides, Saponins, and Anthraquinones are present in all solvents. Flavonoids, Phenols,

and Xanthoproteins are present in Methanol. Alkaloids are present in N-Hexane, Ethyl
 36

acetate and Methanol. Proteins are present in N-Hexane, Ethyl acetate and 50% Aqueous

Methanol. Carbohydrates are present in Methanol and 50% Aqueous Methanol. Resins are

present in N-Hexane. Xanthoproteins are present in Methanol. Oxylates and Tannins are

present in Ethyl acetate and Methanol.

3. The Physicochemical characteristics of the various extracts from Elaeagnus

triflora

3.1 Organoleptic Evaluation

Table 5
Organoleptic Result of Elaeagnus triflora in Leaves
Parameters N-hexane Ethyl acetate Methanol 50% Methanol
Test
Color Yellowish brown Dark green Dark greenish Dark brown
brown
Odor Alcoholic odor Acetone-like odor Acidic odor Acidic odor
Appearance Viscous extract Viscous extract Viscous extract Slightly viscous
extract

The leaf extract residue of n-hexane is viscous, yellowish brown and has alcohol-

like odor; the Ethyl Acetate residue is viscous, dark green and has an acetone-like odor;

the methanol residue is viscous, dark greenish brown and has an acidic odor; and the 50%

aqueous methanol residue is slightly viscous, dark brown and has an acidic odor.

Table 6
Organoleptic Result of Elaeagnus triflora in Stem
Parameters N-hexane Ethyl acetate Methanol 50% Methanol
Test
Color Yellowish brown Greenish brown Dark brown Light brown
Odor Alcoholic odor Acetone-like odor Acidic odor Acidic odor
Appearance Viscous extract Viscous extract Viscous extract Slightly viscous
extract
 37

The stem extract residue of n-hexane is viscous, yellowish brown and has alcohol-

like odor; the Ethyl Acetate residue is viscous, greenish brown and has an acetone-like

odor; the methanol residue is viscous, dark brown and has an acidic odor; and the 50%

aqueous methanol residue is slightly viscous, light brown and has an acidic odor.

Table 7
Organoleptic Result of Elaeagnus triflora in Bark
Parameters N-hexane Ethyl acetate Methanol 50% Methanol
Test
Color Yellowish Dark Dark greenish Dark brown Dark brown
brown brown
Odor Alcoholic odor Acetone-like odor Acidic odor Acidic odor
Appearance Slightly viscous Viscous extract Viscous extract Slightly viscous
extract extract

The bark extract residue of n-hexane is slightly viscous, yellowish dark brown and

has alcohol-like odor; the Ethyl Acetate residue is viscous, dark greenish brown and has an

acetone-like odor; the methanol residue is viscous, dark brown and has an acidic odor; and

the 50% aqueous methanol residue is slightly viscous, dark brown and has an acidic odor.

3.2 Solubility Test

Table 8
Solubility Result of the E.triflora leaves in various extractives
Extracts prepared in Distilled water 5% NaOH 5% NaHCO3 5% HCl
n-hexane Soluble Soluble Soluble Soluble
Ethyl acetate Soluble Insoluble Soluble Insoluble
 38

Methanol Soluble Insoluble Soluble Insoluble

50% Methanol Soluble Soluble Soluble Soluble

The n-hexane and 50% aqueous methanol extract was soluble in all four solvents;

while the ethyl acetate and methanol was soluble in distilled water and 5% NaHCO3.

Table 9
Solubility Result of the E.triflora stem in various extractives
Extracts prepared in Distilled water 5% NaOH 5% NaHCO3 5% HCl
n-hexane Soluble Soluble Soluble Soluble
Ethyl acetate Soluble Insoluble Insoluble Soluble
Methanol Soluble Insoluble Soluble Insoluble
50% Methanol Soluble Soluble Soluble Soluble

The n-hexane and 50% aqueous methanol extract was soluble in all four solvents;

the ethyl acetate was soluble in distilled water and 5% HCl; the methanol was soluble in

distilled water and 5% NaHCO3.

Table 10
Solubility Result of the E.triflora bark in various extractives
Extracts prepared in Distilled water 5% NaOH 5% NaHCO3 5% HCl
n-hexane Insoluble Soluble Soluble Soluble
Ethyl acetate Soluble Insoluble Insoluble Soluble
Methanol Soluble Insoluble Soluble Insoluble
 39

50% Methanol Soluble Soluble Soluble Soluble

The n-hexane extract was soluble in 5%NaOH, 5% NaHCO3, and 5% HCl; the ethyl

acetate was soluble in distilled water and 5% HCl; the methanol was soluble in distilled

water and 5% NaHCO3 and the 50% aqueous methanol extract was soluble in all four

solvents.

3.3. FTIR Analysis

All twelve samples underwent FTIR screening the solvent and part which

shown the presence of flavonoidal groups are found in: Ethyl acetate leaves the functional

groups of flavonoids aromatics are detected at 1590cm-1 and carboxylic acids at 940cm-1.

In methanol leaves are detected in carboxylic acids at 2850cm-1 and ketones at 1715cm-1.

In Ethyl Acetate stem found in esters at 1735cm-1, amines at 1090cm-1, and nitrogen

aliphatic group at 970cm-1. In methanol stem found in carboxylic acid at 1030cm-1. In 50%

aqueous methanol stem are found in amides at 1640cm-1. In n-hexane bark are found in

carboxylic acid at 1570cm-1. In Ethyl Acetate bark found in amides at 1740cm-1. In

methanol bark are found in carboxylic acids at 2850cm-1. And also in 50% aqueous

methanol bark found in Amides at 1635cm-1. The detailed FTIR results may be found in

Appendix.

4. Determination of Antioxidant Property based on Antioxidant Assay

The test was performed using spectrophotometer to measure the absorbance of the

different concentrations of the various extracts in various menstruum. The free radical used
 40

was DPPH and FRAP which aids in estimating the antioxidant activity of the sample as

compared to Ascorbic acid which serves as positive control.

4.1. FRAP Assay

100mcg/mL

50mcg/mL
Concentration

50% Methanol
(ug/ml)

20mcg/mL Methanol
Ethyl acetate
10mcg/mL n-hexane
Ascorbic acid
5mcg/mL

0 0.5 1 1.5 2
Absorbance

Figure 3. Result of antioxidant capacity in various extractives by evaluation of

absorbance intervals in the leaves of E.triflora using FRAP assay

Antioxidant capacity of the extractives was determined by FRAP assay

(Figure 3). In this assay, ferric ions are reduced to ferrous ions in the presence of an

antioxidant (or a reducing agent). The methanol extract in 5mcg/mL (0.207), the 50%

aqueous methanol extract in 10mcg/mL (0.225), the ethyl acetate extract in 20mcg/mL

(0.228), the n-hexane extract in 50mcg/mL (0.240) and 50% aqueous methanol extract in

100mcg/mL (0.307) displayed outmost level of absorbance and antioxidant capacity.

 41

100mcg/mL

50mcg/mL
Concentration

50% Methanol
(ug/ml)

20mcg/mL Methanol
Ethyl acetate
10mcg/mL n-hexane
Ascorbic acid
5mcg/mL

0 0.5 1 1.5 2
Absorbance

Figure 4. Result of antioxidant capacity in various extractives by evaluation of

absorbance intervals in the stem of E.triflora using FRAP assay

Antioxidant capacity of the extractives was determined by FRAP assay (Figure 4).

In this assay, ferric ions are reduced to ferrous ions in the presence of an antioxidant (or a

reducing agent). The n-hexane extract in 5mcg/mL (0.229), the 50% aqueous methanol

extract in 10mcg/mL (0.228), the 50% aqueous methanol extract in 20mcg/mL (0.242), the

50% aqueous methanol extract in 50mcg/mL (0.424), the ethyl acetate extract in

100mcg/mL (0.280) displayed outmost level of absorbance and antioxidant capacity.

 42

100mcg/mL

50mcg/mL
Concentration

50% Methanol
(ug/ml)

20mcg/mL Methanol
Ethyl acetate
10mcg/mL
n-hexane
5mcg/mL Ascorbic acid

0 0.5 1 1.5 2
Absorbance

Figure 5. Result of antioxidant capacity in various extractives by evaluation of

absorbance intervals in the bark of E.triflora using FRAP assay

Antioxidant capacity of the extractives was determined by FRAP assay (Figure 5).

In this assay, ferric ions are reduced to ferrous ions in the presence of an antioxidant (or a

reducing agent). The n-hexane extract in 5mcg/mL (0.254), 20mcg/mL (0.247) 50mcg/mL

(0.250) and 100mcg/mL (0.296) and the methanol extract in 10mcg/mL (0.310) displayed

outmost level of absorbance and antioxidant capacity.

4.2. DPPH Assay

100mcg/mL
Concentration

50mcg/mL
(ug/ml)

50% Methanol
20mcg/mL Methanol
Ethyl acetate
10mcg/mL
n-hexane
5mcg/mL Ascorbic acid

0 0.5 1 1.5 2
Absorbance

Figure 6. Result of antioxidant capacity in various extractives by evaluation of

absorbance intervals in the leaves of E.triflora using DPPH assay
 43

Free radical scavenging effects of various extractives used in the leaves of E.

triflora at different concentrations were measured with ascorbic acid as standard compound

by using the DPPH method. The methanol extract in 5mcg/mL (1.581), the n-hexane

extract in 10mcg/mL (1.621), the methanol extract in 20mcg/mL (1.634), the n-hexane

extract in 50mcg/mL (1.618), the n-hexane extract in 100mcg/mL (1.619) displayed

outmost level of absorbance and antioxidant capacity.

100mcg/mL
Concentration

50mcg/mL
(ug/ml)

50% Methanol
20mcg/mL Methanol
Ethyl acetate
10mcg/mL
n-hexane
5mcg/mL Ascorbic acid

0 0.5 1 1.5 2
Absorbance
Figure 7. Result of antioxidant capacity in various extractives by evaluation of
absorbance intervals in the stem of E.triflora using DPPH assay

Free radical scavenging effects of various extractives used in the stem of E. triflora

at different concentrations were measured with ascorbic acid as standard compound by

using the DPPH method. The 50% aqueous methanol extract in 5mcg/mL (1.619), the

methanol extract in 10mcg/mL (1.620), the n-hexane extract in 20mcg/mL (1.594), the

methanol extract in 50mcg/mL (1.637), the n-hexane extract in 100mcg/mL (1.646)

displayed outmost level of absorbance and antioxidant capacity.

 44

100mcg/mL

50mcg/mL
Concentration

50% Methanol
(ug/ml)

20mcg/mL Methanol
Ethyl acetate
10mcg/mL
n-hexane

5mcg/mL Ascorbic acid

0 0.5 1 1.5 2
Absorbance

Figure 8. Result of antioxidant capacity in various extractives by evaluation of

absorbance intervals in the bark of E.triflora using DPPH assay

Free radical scavenging effects of various extractives used in the bark of E. triflora

at different concentrations were measured with ascorbic acid as standard compound by

using the DPPH method. The 50% aqueous methanol extract in 5mcg/mL (1.619), the n-

hexane extract in 10mcg/mL (1.608), methanol extract in 20mcg/mL (1.638), the n-hexane

extract in 50mcg/mL (1.644), the 50% aqueous methanol extract in 100mcg/mL (1.682)

displayed outmost level of absorbance and antioxidant capacity.

 45

CHAPTER 5

Summary, Conclusions and Recommendations

This chapter summarizes the results of Phytochemical Screening and

determination of antioxidant property of the crude extract from leaves, stem, and bark of

Elaeagnus triflora, family Elaeagnaceae. The conclusion and recommendation of the

researchers are hereby presented.

Summary of Findings

Based on the research objectives, the summaries of findings are hereby presented:

1. The dried leaves, stem and bark of E.triflora were collected from Mabacan,

Calauan, Laguna during the month of July 2018 and was authenticated by UP

Biology, Institute of Biology, College of Science in Diliman, Manila. The dried

cut leaves, stem and bark underwent serial exhaustive extraction and were

macerated in series using n-Hexane, Ethyl acetate, Methanol and 50% Aqueous

Methanol respectively. The residue was weighed and the percentage yield was

noted as; [1] n-Hexane, [2] Ethyl acetate, [3] Methanol and [4] (50%) Aqueous

Methanol chronologically, evaporated to syrupy consistency and was weighed

to get the percentage yield. The percentage yield of [1] n-hexane; (a) Leaves was

9.54 % (b) Stem was 7.37% (c) Bark was 7.91% [2] Ethyl acetate; (a) Leaves

was 5.21% (b) Stem was 7.29% (c) Bark was 7.39 % [3] Methanol; (a) Leaves

was 13.15% (b) Stem was 10.42% (c) Bark was 9.66% [4] (50%) Aqueous

Methanol (a) Leaves was 6.69% (b) Stem was 4.41% (c) Bark was 10.22%.
 46

2. Phytochemical analysis of leaf, stem and bark extracts (n-hexane, ethyl acetate,

methanol and 50% aqueous methanol) showed the presence of terpenoids,

alkaloids, glycosides, saponins, anthraquinones and oxylates in all extracts.

Flavonoids and phenol were found to be present only in methanolic extract of the

bark. Tannins were only present in the ethyl acetate extract of the leaves, stem in

50% aqueous methanol, bark in ethyl acetate and methanol solvent. Carbohydrates

were only present in leaves, stem and bark under methanol and 50% aqueous

methanol. Xanthoproteins were only present in leaves and bark under methanol

solvent. Resins, lipids, phlobatannins, quinines were found to be absent in all tested

extracts.

3. The leaf extract residue of n-hexane is viscous, yellowish brown and has

alcohol-like odor; the Ethyl Acetate residue is viscous, dark green and has an

acetone-like odor; the methanol residue is viscous, dark greenish brown and has an

acidic odor; and the 50% aqueous methanol residue is slightly viscous, dark brown

and has an acidic odor.

4. Using four reagents in solubility test namely distilled water, 5%NaOH,

5%NaHCO3 and 5%HCl in leaves, stem and bark in different solvents the

produced results were n-hexane and 50% aqueous methanol leaves and stem were

all soluble in all solvents while in bark, 50% aqueous methanol are all soluble. N-

hexane bark extracts are all soluble except in distilled water. Ethyl acetate and

methanol leaves extracts are soluble in distilled water and 5% NaHCO3 while

insoluble in 5%NaOH and 5% HCl. Methanol stem and bark were soluble in
 47

distilled water and 5%NaHCO3, insoluble in 5%NaOH and 5%HCl. Ethyl acetate

stem and bark were soluble in distilled water and 5%HCl but insoluble in 5%NaOH

and 5%NaHCO3.

5. The functional groups present in the leaves extract in n-hexane are mostly

consisting of alkanes, ethyl acetate contains alkanes, amides, ethers, alcohols,

phenols and carboxylic acids, methanol contains phenols, carboxylic acids, ketones

and alkenes, 50% aqueous methanol contains alkenes.

6. From the result of FRAP assay the bark part under produces the highest level of

antioxidant activity in 5mcg/ml, 20mcg/ml in n-hexane, 10mcg/ml in methanol,

50mcg/ml in aqueous methanol, and 100mcg/ml leaves antioxidant also is present.

7. The result from the DPPH assay in various extractives in 10mcg/ml, 20mcg/ml,

50mcg/ml and 100mcg/ml evaluation of its variable with the positive control was

determined. In 5mcg/ml methanol extract in leaves gave the closest number value

to the positive control. The n-hexane extract in leaves, stem and bark used in DPPH

assay mostly shown close number values to the positive control and the 2nd closely

was the methanol extract in the leaves part.

8. The 5mcg/ml ethyl acetate extract in bark and 10mcg/ml concentration of ethyl

acetate extract in leaves and stem were the most effective in the antioxidant activity

as indicated by the percentage scavenging activity for DPPH assay reading in

figure 3,4 and 5. The other concentrations are deemed ineffective than the positive

control or just outright lacking in terms of eliciting an antioxidant property.

Conclusion
 48

Based on the findings, the study has shown that the leaves, stem and bark of E. triflora

has the following phytoconstituents: terpenoids, alkaloids, glycosides, saponins,

anthraquinones and oxylates. E.triflora exhibited low antioxidant property as compared

with the standard.

Recommendation

1. Further works and studies may be performed on the isolation and identification of the

concrete antioxidant components present in Elaeagnus triflora.

2. E.triflora elicits alkaloidal property which can be utilized for further studies such as

antiproliferative and antidepressant activity.

3. Determination of diuretics and carditonic activity of E.triflora must be conducted since

it contains glycosides.

4. Further studies related to pesticides and insecticides usage of this plant must be obtained

since the plant gains anthraquinones property.

5. E.triflora claims oxylates property which is considered as a toxic substance, therefore,

we advise other researchers to establish a well safety profile.

6. It is advised that other parts of the plant such as roots and flowers should be used to

determine antioxidant property.